High-throughput functional genomics using CRISPR-Cas9

By Ophir Shalem, Neville E. Sanjana, Feng Zhang. Nature reviews genetics, 2015

1,168 citations recorded by the source corpus.

Forward genetic screens are powerful tools for the discovery and functional annotation of genetic elements. Recently, the RNA-guided CRISPR (clustered regularly interspaced short palindromic repeat)-associated Cas9 nuclease has been combined with genome-scale guide RNA libraries for unbiased, phenotypic screening. In this Review, we describe recent advances using Cas9 for genome-scale screens, including knockout approaches that inactivate genomic loci and strategies that modulate transcriptional activity. We discuss practical aspects of screen design, provide comparisons with RNA interference (RNAi) screening, and outline future applications and challenges.

Subfields: Molecular Biology

View Paper: https://www.semanticscholar.org/paper/703fd40cf1fe71857970b3fc7e09b9032a6ac3a4

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Citations in the corpus

Compact and highly active next-generation libraries for CRISPR-mediated gene repression and activation (2016, background): “Recently, a number of pooled screening platforms have been developed that utilize the bacterial CRISPR (Clustered Regularly Interspaced Palindromic Repeats)-associated nuclease Cas9 paired with libraries of single-guide RNAs (sgRNAs) to disrupt targeted genes (reviewed in Shalem et al., 2015).”
The WRKY Transcription Factor Family in Model Plants and Crops (2017, background): “Relying on these convenient characters, some CRISPR/Cas9 mutant libraries have been developed for genome-wide mutation screens in cultured eukaryotic cells (Shalem et al., 2015).”
Next-Generation Sequencing — An Overview of the History, Tools, and “Omic” Applications (2016, background): “Moreover, the CRISPR/CAS systems have been developed as an “in vitro” genetic engineering tool to be transfected into the cells of various organisms to manipulate their genes [196], including the foreign defense system introduced into human cells against HIV-1 infection [197].”
Applications of genome editing technology in the targeted therapy of human diseases: mechanisms, advances and prospects (2020, methodology): “screens, the BRAF inhibitor vemurafenib was used to treat a genome-scale knockout library of melanoma cells and recovered genes conferring resistance to the drug.(253) Similar to the CDKO system, another simple and effective strategy for analyzing the function of combinatorial genes is CombiGEM-CRIS”
The BioGRID interaction database: 2019 update (2018, background): “Cas9 expressed in cell lines can be programmed with a complex library pool of single-guide RNAs (referred to as sgRNAs or gRNAs) to efficiently generate double strand breaks at targeted loci across the genome and thereby yield a pool of loss-offunction mutants due to error prone repair of the break;”
The BioGRID database: A comprehensive biomedical resource of curated protein, genetic, and chemical interactions (2020, methodology): “48 Programmable CRISPR-based endonucleases in conjunction with genome-wide pools of single guide RNAs (sgRNAs) that direct the nuclease to specific sites in the genome have revolutionized genetic screens in human cell lines and other animal model systems.”
High-Content Screening for Quantitative Cell Biology. (2016, background): “OneofthemostpromisingisCRISPR/Cas9mediated(multi-)geneediting[62],whichcanbe used for the large-scale construction of gene mutations or collections of tagged genes in mammalian and other eukaryotic cells.”
Genome-wide Programmable Transcriptional Memory by CRISPR-based Epigenome Editing (2021, methodology): “CRISPRoff provides a valuable complement to existing CRISPRi and CRISPR nuclease approaches (Doench, 2018; Hanna and Doench, 2020; Shalem et al., 2015).”
Single-Cell and Single-Molecule Analysis of Gene Expression Regulation (2016, background): “Also, recent advances in CRISPR-Cas technology have greatly facilitated genome editing (129) and RNA labeling (108) for live cell imaging.”
Computer vision for high content screening (2016, background): “In functional genomics screens, cells with different genetic perturbations (such as a genetic deletion or RNA interference) are arrayed and imaged to discover functions and relationships between genes (Carpenter & Sabatini, 2004; Houle et al ., 2010; Shalem et al ., 2015).”